Biocidal polymers

ABSTRACT

Pharmaceutical compositions containing biocidal co-polymers of poly(styrenes), poly(acrylates), poly(acrylamides), and poly(C 1 -C 6 )alkylene glycols are disclosed, along with methods of using the compositions to treat microbial infections in mammals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/933,987, filed 3 Sep. 2004, which claims priority under 35 USC§119(e) to U.S. Provisional Application Ser. No. 60/500,201, filed 4Sep. 2003, the entireties of both of which are incorporated herein byreference.

FEDERAL FUNDING

This invention was made with government support under 0140621 awarded bythe National Science Foundation. The government has certain rights inthis invention.

INCORPORATION BY REFERENCE

All of the references cited below are incorporated herein by reference.

FIELD OF THE INVENTION

The invention is directed to pharmaceutical compositions containingbiocidal co-polymers of poly(styrenes), poly(acrylates),poly(acrylamides), and poly(C₁-C₆)alkylene glycols. More specifically,the invention is directed to pharmaceutical compositions and methods oftreating microbial infections in mammals.

BACKGROUND

The emerging prevalence of bacteria resistant to common therapeuticagents has led to a dire need for new antimicrobial compounds.¹ Peptideantimicrobials,² a central element of the human immune system, havereceived increasing interest as potentially new antimicrobialtreatments. One reason for their potential success is that it appears tobe difficult, although not impossible, for pathogenic microbes todevelop resistance to these innate “host-defense” peptides. One largesubset of host-defense peptides forms an amphiphilic α-helicalstructure.³ These peptides act by disrupting bacterial membranes becausetheir net positive charge attracts the peptides to the negativelycharged bacterial membrane,⁴ and the hydrophobic face of the helixallows the formation of aggregates that compromise membrane integrity.²Amphiphilic topology also plays a factor in the biological activity ofthese molecules, as enantiomeric peptides retain full activity.⁵

This design principle has been applied to distinct types of amphiphilichelical antimicrobial oligomers. For example, β-Amino acid oligomers(“β-peptides”) can adopt discrete helical conformations.⁶ By properlyarranging cationic and lipophilic residues within the β-peptide,amphiphilic helices with antimicrobial activity can by obtained.⁷⁻⁹DeGrado et al. also describe aryl amide oligomers with elongatedconformations that can project lipophilic and cationic groups toopposite sides of the molecular backbone.¹⁰ Unlike α- or β-peptideoligomers, these aryl amide oligomers are achiral.

SUMMARY OF THE INVENTION

The invention disclosed and claimed herein arose from the inventors'interest in determining how much conformational “pre-organization” isrequired for antimicrobial activity in synthetic oligomers or polymers.For example, the helical, antimicrobial β-peptides developed by Gellmanet al. are composed of cyclically-constrained β-amino acids and arequite rigid.^(8,9) As a result, antimicrobial activity is generallyattenuated or lost entirely upon sequence scrambling to give anon-amphiphilic helix.⁹ In contrast, α-helical host-defense peptidescomposed of α-amino acid residues can be scrambled with only modest lossof activity.¹¹ This difference in the effect of residue scrambling canbe explained by invoking the increased flexibility of the α-peptidebackbone, which might allow scrambled sequences to populate non-helical,but globally amphiphilic, conformations. The highly preorganizedβ-peptide backbone cannot adopt such non-helical conformations. Whilethis mechanistic theory appears to explain the difference in activityobserved between α-peptides and β-peptides, Applicants are not limitingtheir invention to any specific mechanistic pathway.

Extending this hypothesis, the present inventors postulate that anysufficiently flexible synthetic polymer backbone might display a randomsequence of cationic and lipophilic side-chains in a manner that resultsin global amphiphilicity and, therefore, biocidal activity in generaland antimicrobial activity in particular. This hypothesis was put to thetest by directly comparing cationic polystyrene-based polymers to anα-peptide known to display potent antimicrobial activity. Polystyreneswere chosen as a test backbone. Other polymeric species, such aspoly(alkylene glycols) (e.g., poly(ethylene glycol)) and poly(acrylates)are also included within the scope of the present invention. Polystyrenederivatives are preferred.

The present invention, therefore, is drawn to a new class of biocidaland antimicrobial polymers in which a poly(styrene), a poly(acrylamide),a poly(acrylate), or a poly(C₁-C₆-alkylene glycol) backbone is combinedwith side-chains that require protonation for positive charge. Theresults represent the first direct comparison between an antimicrobialα-peptide and synthetic polymers. As shown by the Examples presentedbelow, styrene copolymers can display biocidal activity comparable tothat of a modified host-defense peptide. This result supports thehypothesis that backbone “pre-organization” is not essential for potentbiocidal and antimicrobial activity.²⁷

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions: The following abbreviations are usedthrough the specification and claims:

AIBN=2,2′-azo-bis-isobutyrylnitrile, a free-radical polymerizationinitiator

ANS=the dye 4-aminonaphthalene-1-sulphonic acid

BHI=brain-heart infusion broth

DEPT-135=Distortionless Enhancement by Polarization Transfer. In ¹³CNMR, the DEPT technique enables carbon atoms to be assigned according tothe number of attached protons. The DEPT-135 technique shows —CH3 and—CH— carbons as having positive phase and —CH₂— carbons as havingnegative phase. Quaternary carbons do not appear in the DEPT technique.See, for example, Doddrell, et al.²⁸

DMAS=4-(dimethylaminomethyl)-styrene (compound 1)

GPC=gel permeation chromatography

Initiator=a compound capable of initiating free-radical polymerization.A host of initiators are known in the art and include azo compounds(e.g., AIBN), dialkyl and diacyl peroxides, hydroperoxides, peresters,and organic polyoxides. AIBN is preferred. Initiators are widelyavailable commercially, such as from DuPont (marketed as “VAZO”-brandfree radical initiators, Wilmington, Del.) and from Aldrich FineChemicals (Milwaukee, Wis.). Exemplary initiators include, withoutlimitation, t-amyl peroxybenzoate, 4,4-azo-bis-(4-cyanovaleric acid),1,1′-azo-bis-(cyclohexanecarbonitrile), AIBN, benzoyl peroxide,2,2-bis-(t-butylperoxy)butane, 1,1-bis-(t-butylperoxy)cyclohexane,2,5-bis-(t-butylperoxy)-2,5-dimethylhexane,2,5-bis-(t-butylperoxy)-2,5-dimethyl-3-hexyne, t-butyl-peracetate,t-butyl peroxide, t-butyl peroxybenzoate, t-butylperoxy-isopropylcarbonate, cumene hydroperoxide, peracetic acid, potassium persulfate,and the like. See Denisov et al.²⁹ for an exhaustive treatment offree-radical initiators and free-radical-mediated polymerizations.

MBC=minimal bactericidal concentration; the minimum concentration of anactive agent that kills all or substantially all of the selected targetcell type.

MIC=minimum inhibitory concentration; the minimum concentration of anactive agent that inhibits growth of the selected target cell type.

NMR=nuclear magnetic resonance spectroscopy

PDI=polydispersity index

Pharmaceutically-suitable salt: any salt conventionally used in theformulation of pharmaceutical compositions for ingestion, injection, ortopically application, including, without limitation, those derived frommineral acids and organic acids, explicitly including hydrohalides,e.g., hydrochlorides and hydrobromides, sulphates, phosphates, nitrates,sulphamates, acetates, citrates, lactates, tartrates, malonates,oxalates, salicylates, propionates, succinates, fumarates, maleates,methylene-bis-b-hydroxynaphthoates, gentisates, isethionates,di-p-toluoyltartrates, methane-sulphonates, ethanesulphonates,benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates,quinates, and the like; and base addition salts, including those derivedfrom alkali or alkaline earth metal bases or conventional organic bases,such as triethylamine, pyridine, piperidine, morpholine,N-methylmorpholine, and the like.

UV-Vis=ultraviolet-visible spectroscopy

Biocidal and Antimicrobial Pharmaceutical Compositions:

The biocidal and antimicrobial compositions of the present inventioncontain as active ingredients co-polymers of poly(styrenes),poly(acrylates), poly(acrylamides), and poly(C₁-C₆-alkylene glycols).Co-polymers of poly(styrenes) are preferred. Copolymers of4-(dimethylaminomethyl)-styrene (1) and 4-octylstyrene (2) were preparedby a free-radical polymerization method.¹² Hydrophobic size-exclusionchromatography using Sephadex LH-20 yielded samples of polymer withoutmonomer vinyl peaks in the ¹H NMR spectrum. For clarity and brevity,copolymers will be referred to herein by the molar percentages ofmonomers in the feed mixture. Thus, copoly(1₉₅:2₅) refers to the productgenerated by polymerization of a mixture of 95 mol % 1 and 5 mol % 2.Analysis of the copolymers indicated that their composition closelyapproximates that of the feed mixture.¹³

Polymer molecular weight averages were determined by GPC usingliterature methods.¹⁴ Most polymers had molecular weights (Mw) close to9000 Da, with polydispersity indices, PDI's (i.e., Mw/Mn) close to 3.0,which is typical for AIBN-initiated radical polymerizations. These datayield Mn values near 3000, which are comparable to the molecular weightof the magainin derivative used as a standard (MW=2478). Achromatographic assay demonstrated that these materials were in factcopolymers of 1 and 2 and not simply mixtures of polymers formed fromthe homopolymerization of 1 and 2.¹⁴

Thus, the present invention is directed to biocidal and antimicrobialpharmaceutical compositions (homopolymers and heteropolymers), whereinthe active ingredient comprises one or more polymers of the formula-(A)_(n)-, wherein each A is a residue independently selected from thegroup consisting of:

wherein each R is independently selected from the group consisting ofhydrogen, linear or branched C₁-C₃₀-alkyl, alkenyl, or alkynyl,unsubustituted amino-C₁-C₆-alkyl, mono-substituted amino-C₁-C₆-alkyl,disubstituted amino-C₁-C₆-alkyl, mono- or bicyclic aryl, mono- orbicyclic heteroaryl having up to 5 heteroatoms selected from N, O, andS; mono- or bicyclic aryl-C₁-C₆-alkyl, mono- or bicyclicheteroaryl-C₁-C₆-alkyl, —(CH₂)_(n+1)—OR¹, —(CH₂)_(n+1)—SR¹,—(CH₂)_(n+1)—S(═O)—CH₂—R¹, —(CH₂)_(n+1)—S(═O)₂—CH₂—R¹,—(CH₂)_(n+1)—NR¹R¹, —(CH₂)_(n+1)—NHC(═O)R1,—(CH₂)_(n+1)—NHS(═O)₂—CH₂—R¹, —(CH₂)_(n+1)—O—(CH₂)_(m)—R²,—(CH₂)_(n+1)—S—(CH₂)_(m)—R², —(CH₂)_(n+1)—S(═O)—(CH₂)_(m)—R²,—(CH₂)_(n+1)—S(═O)₂—(CH₂)_(m)—R², —(CH₂)_(n+1)—NH—(CH₂)_(m)—R²,—(CH₂)_(n+1)—N—{(CH₂)_(m)—R²}₂, —(CH₂)_(n+1)—NHC(═O)—(CH₂)_(n+1)—R², and—(CH₂)_(n+1)—NHS(═O)₂—(CH₂)_(m)—R²;

wherein R¹ is independently selected from the group consisting ofhydrogen, C₁-C₆-alkyl, alkenyl, or alkynyl; mono- or bicyclic aryl,mono- or bicyclic heteraryl having up to 5 heteroatoms selected from N,O, and S; mono- or bicyclic aryl-C₁-C₆-alkyl, mono- or bicyclicheteroaryl-C₁-C₆-alkyl; and

wherein R² is independently selected from the group consisting ofhydroxy, C₁-C₆-alkyloxy, aryloxy, heteroaryloxy, thio, C₁-C₆-alkylthio,C₁-C₆-alkylsulfinyl, C₁-C₆-alkylsulfonyl, arylthio, arylsulfinyl,arylsulfonyl, heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl,amino, mono- or di-C₁-C₆-alkylamino, mono- or diarylamino, mono- ordiheteroarylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,N-aryl-N-heteroarylamino, aryl-C₁-C₆-alkylamino, carboxylic acid,carboxamide, mono- or di-C₁-C₆-alkylcarboxamide, mono- ordiarylcarboxamide, mono- or diheteroarylcarboxamide,N-alkyl-N-arylcarboxamide, N-alkyl-N-heteroarylcarboxamide,N-aryl-N-heteroarylcarboxamide, sulfonic acid, sulfonamide, mono- ordi-C₁-C₆-alkylsulfonamide, mono- or diarylsulfonamide, mono- ordiheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide, urea;mono- di- or tri-substituted urea, wherein the subsitutent(s) isselected from the group consisting of C₁-C₆-alkyl, aryl, heteroaryl;O-alkylurethane, O-arylurethane, and O-heteroarylurethane;

wherein R′ is selected from same group recited above for R, providedthat one of R′ is hydrogen;

wherein n is an integer ≧3 (preferably ≧6);

provided that at least one “A” is a nitrogen-containing residue whereinthe nitrogen atom is capable of being protonated;

and pharmaceutically-suitable salts thereof; in combination with apharmaceutically-suitable carrier.

Note that where a substituent is designated as being “independentlyselected” from a given set of moieties, each appearance of the statedsubstituent can be different. Thus, for example, the formula -(A)_(n)-comprises homopolymers wherein each appearance of “A” is the same, andheteropolymers where each appearance of “A” is different (i.e.,A-A′-A′″-A′″). The same applies for the various R substituents.

Synthesis of the poly(styrene), poly(acrylamide), and poly(acrylate)co-polymers according to the present invention is preferably carried outvia free-radical-mediated polymerization, as described in the Examples.

While poly(C₁-C₆-alkylene glycol) co-polymers can by synthesized usingfree-radical methods, it is not the preferred route. Using poly(ethyleneglycol) (PEG) as an example, PEG is preferably synthesized via aring-opening reaction of ethylene oxide, which is widely known in theart. Poly(alkylene glycol) can be co-polymerized with the other monomertypes described herein using the methods described in, for example,Ishizu, Shen & Tsubaki (March 2000) Polymer, 41(6):2053-2057, and Cheng,Wang & Chen (February 2003) Materials Chemistry & Physics 78(3):581-590.For methods of synthesizing co-polymers of PEG and poly(acrylamides),see Auzanneau et al. (1998) Can. J. Chem. 76(8):1109-1118.

Ring-opening polymerizations of epoxides, such as ethylene oxide orpropylene oxide may be accomplished using cationic ring-openingpolymerization. Strong protic acids are conventionally used as acatalyst, e.g., H₂SO₄, CF₃SO₃H, and CF₃CO₂H. The same polymerization canalso be accomplished using anionic ring-opening polymerization. Typicalinitiators for anionic ring-opening polymerization include, withoutlimitation, alkali metals (Na, K), inorganic bases (NaOH, KOH), metaloxides (LiOCH₃), and metal alkyls and hydrides (BuLi, NaH).

Polymerization can be head-to-tail and/or head-to-head, syndiotactic,isotactic, and combinations thereof.

Polymers that are cationic by virtue of quaternized nitrogen atoms andstructurally related to poly(l) and copoly(1:2) have been studied asantimicrobial agents.¹⁴⁻¹⁸ However, polymers prepared from 1 differsignificantly from the quaternized prior art compounds in that, likehost-defense peptides, polymers containing dimethylaminomethyl groupsrequire protonation to develop positive charge. Thus, the preferredcompounds for use in the present invention are those containing afraction (e.g., from about 1 mol % to about 99 mol %) of R groups thatrequire protonation to develop positive charge, and a fraction that or Rgroups (e.g., from about 99 mol % to about 1 mol %) that are neutral oranionic.

Two examples from the quaternized class of compounds were used forcomparison with the protonatable polymers described herein. Poly(5) wassynthesized from monomer 5 via reported methods.^(16,19) Poly(7) waspurchased (Sigma, St. Louis, Mo.) in a form that is >98% quaternized,having an Mn=12.0 KDa and a PDI=1.06. Both poly(5) and poly(7) arequaternized via N-methylation. These polymers have shorter alkyl chainsthan the octyl group of (2) but are preferred for this work becauselimited aqueous solubility was reported by Senuma et al. for theN-dodecyl analogue of poly(5)¹⁸ and by Lin et al. for the N-hexylanalogue of poly(7).²⁰

Minimum inhibitory concentrations (MICs) for poly(1) and the copoly(1:2)series were determined against E. coli, ²¹ B. subtilis, ²²methicillin-resistant S. aureus ²³ (MRSA), and vancomycin-resistant E.faecium ²⁴ (VRE). Assays were conducted at polymer concentrations up to50 μg/mL. Most polymers were not tested at higher concentrations due tolimited solubility in the assay media. The data given are conservativeestimates of MIC because some precipitation occurred (at concentrationsabove 12.5 μg/mL) over the 6-hour incubation period, thus causingturbidity even in the absence of bacteria. A high-activity magaininanalogue, (Ala^(8,3,18))-magainin-2-amide,²⁵ was used as a positivecontrol.

The tertiary amine-containing polymers show inhibitory activity similarto that of the magainin against all four organisms tested. Monomer 1showed no growth inhibition. Poly(5) was inactive in all experimentsexcept one, inhibiting B. subtilis at 50 μg/mL. Past assays of poly(5)used agar plate assays,^(15,18,19) which are prone to artifacts due tointeraction of the polymers with the agar,¹⁶ or cell viability countingafter incubating cells with polymer in sterile water or saline.¹⁶ Thepoor activity of poly(5) in our brain-heart infusion broth (BHI) growthinhibition assay could be due to interaction with anionic components ofthe broth.¹⁶ Methylpyridinium-bearing poly(7) showed some inhibition forthree strains. This moderate activity is comparable to that observed byLin et al. for poly(7) of tenfold higher molecular weight.²⁰

TABLE 1 Minimum Inhibitory Concentrations (MIC's, (μg/mL) for polymersvs. the four bacterial strains tested. Subscripts refer to mole fractionof monomer. E. coli B. subtilis S. aureus E. faecium Strain JM109 BR1515332 A436 poly(1) 25 12.5 50 12.5 Copoly(1₉₇:2₃) 50 12.5 25 6.3Copoly(1₉₅:2₅) 50 12.5 25 6.3 copoly(1₉₃:2₇) 25 6.3 12.5 3.2copoly(1₉₀:2₁₀) 50 12.5 17.8 6.3 copoly(1₈₅:2₁₅) 50 25 25 12.5copoly(1₈₀:2₂₀) 50 35.4 25 25 copoly(1₇₀:2₃₀) >50 50 >50 50copoly(1₆₅:2₃₅) >50 >50 >50 50 (Ala^(8,3,18))- 12.5 6.3 12.5 3.2magainin-2-amide poly(5) >50 50 >50 >50 poly(7) >50 25 35.4 50 (1)(monomer) >50 >50 >50 >50

Both nonpolar and electrostatic forces are believed to be important inthe interactions of host-defense peptides with bacterial membranes.²⁶Prior studies of copolymers of vinylbenzylammonium^(18,19) andvinylpyridinium¹⁷ salts have shown that antimicrobial activity isinfluenced by the proportion of cationic and lipophilic monomers. In anexamination of N-benzyl-4-vinyl pyridinium/styrene copolymers, Li et al.found that sterilizing activity at first remained constant as theproportion of styrene was raised; once a threshold value was reached,however, additional styrene incorporation led to decreased activity.¹⁷The present data for copoly(1:2) show a related pattern. Below 20-30%(2), antimicrobial activity is only modestly affected by the proportionof lipophilic component (2), but above this level activity dropsprecipitously.

GPC analysis showed that for the polymers described in the Examples, theGPC procedure used gives similar, broad molecular weight distributions,regardless of polymer composition, a phenomenon typical ofAIBN-initiated free-radical polymerizations.

These polymers contain both hydrophobic and hydrophilic functionality.Their hydrophobic portions could be either buried within micelle-likestructures or exposed to solvent. Colorimetric assays, as described inthe Examples, were performed to test each of these hypotheses.Specifically, solubilization of the hydrophobic dye Orange OT canindicate the formation of micelle-like structures with hydrophobicinterior regions. Single polymer molecules might form such structureseven at low concentrations, but the data collected to date (not shown)did not show significant, reproducible dye solubilization. Hence, at theconcentrations studied, the subject polymers do not act as conventionalmicellar detergents.

The dye 4-anilinonaphthalene-1-sulfonic acid (ANS) shows a drasticincrease in fluorescence on binding solvent-exposed hydrophobic patchesin the “molten globule” state of proteins. See Cavagnero et al. (1995)Biochemistry 34:9865-0873, and Semisotnov et al. (1991) Biopolymers31:119-128. Fluorescence also correlates to the amount of exposedhydrophobic surface displayed by poly(amino acid)s. When polymers wereexcited at 350 nm to avoid fluorescence from the aromatic polymersthemselves, significant enhancement of ANS fluorescence was seen,suggesting exposure of hydrophobic surface, but no correlation wasevident with polymer composition.

The data on Orange OT solubilization and ANS fluorescence, takentogether, are consistent with a “molten globule-like” model of polymerconformation. In such a model, these polymers cannot present solelycationic functionality to the solvent surface at the concentrationsstudied.

The tertiary amine-containing polymers show inhibitory activity againstall four organisms tested. Because data points are taken at twofolddilutions, a twofold variation in MIC corresponds to a single datapoint. Therefore, even the apparently wide variation in MIC seen shouldbe interpreted with great caution.

The polymers also show bactericidal activity against both Gram-positiveand Gram-negative pathogens.

The interaction between anionic bacterial membranes and antimicrobialpeptides is believed to be primarily electrostatic. Nevertheless,poly(l) and its derivatives displayed antibacterial activity similar tothat of magainin, whereas poly(5) and poly(6) showed little to noactivity.

Past assays of poly(5) and poly(6) used agar plate assays (which havesince been discredited) or cell viability counting after incubatingcells with polymer in sterile water or saline. The low activity of thecomparison polymers in the BHI growth inhibition assay may be due tointeraction between the polymers and anionic components of the broth.However, a rich medium like BHI may better represent conditionsencountered during infection of a host organism.

All of the tertiary amine polymers described in the Examples are usefulas broad-spectrum biocides and antiseptics.

Pharmaceutical compositions of the present invention, comprise abiocidal- or antimicrobial-effective amount of an active compound asdescribed above, an isomeric form thereof, or a pharmaceuticallyacceptable salt of the compound or isomeric form thereof, together withan acceptable carrier for it, and optionally other therapeuticallyactive ingredients. The carrier must be “pharmaceutically acceptable” or“pharmaceutically suitable,” i.e., the carrier must be compatible withthe other ingredients of the composition and not deleterious to therecipient thereof.

The compositions include those suitable for oral, rectal or parenteral(including subcutaneous, intramuscular, intradermal and intravenous)nasal, or bronchial administration. Topical formulations are alsoincluded, for example, for topical antibiotic use.

It is noted that some of the compounds and isomers described hereinthereof are also rather insoluble in water and, accordingly, liquidformulations which account for this factor may be made according toart-recognized pharmaceutical techniques. Examples of these techniquesinclude an injection wherein the active compound is dissolved in asuitable solvent or co-solvent such as an appropriate polyethyleneglycol, or a propylene glycol or the like; a sealed gelatin capsuleenclosing an oily solution of the active compound; a suppository of theactive compound in a conventional suppository base such as cocoa butter;or a lipo some formulation, for example, the active compound and aglycerophospholipid such as phosphatidylcholine. In any event, theaforementioned characteristics of the subject compounds and isomers arenot uncommon in the pharmaceutical art and, accordingly, art-recognizedpharmaceutical techniques are employed to prepare appropriateformulations for such compounds, isomers or pharmaceutically acceptablesalts of either.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound or saltinto association with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound or salt into association with aliquid or solid carrier and then, if necessary, shaping the product intodesired unit dosage form.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,boluses or lozenges, each containing a predetermined amount of theactive compound (optionally in the form of a salt thereof); as a powderor granules; or in liquid form, e.g., as suspension, solution, syrup,elixir, emulsion, dispersion, or the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine a mixture of the powdered active compound with anysuitable carrier.

Formulations suitable for parenteral administration convenientlycomprise a sterile preparation of the active compound (optionally in theform of a salt thereof) in, for example, a polyethylene glycol 200 orpropylene glycol solution which is preferably isotonic with the blood ofthe recipient.

Useful formulations also comprise concentrated solutions or solidscontaining the active compound(s), any isomeric form thereof, or apharmaceutically acceptable salt of the compound or isomeric formthereof, which upon dilution with an appropriate solvent give a solutionsuitable for parenteral administration.

Preparations for topical or local applications, which are, for example,conventional for preventing or treating bacterial infections of theskin, mouth, and eyes, comprise aerosol sprays, lotions, gels,ointments, etc. and pharmaceutically acceptable vehicles therefore suchas, for example, lower aliphatic alcohols, polyglycerols such asglycerol, polyethyleneglycerol, esters of fatty acids, oils and fats,silicones, and other conventional topical carriers.

In topical formulations, the active compounds (or isomers thereof) arepreferably utilized at concentrations of from about 0.1% to about 5.0%percent by weight.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more optional accessoryingredients(s) utilized in the art of pharmaceutical formulations, e.g.,diluents, buffers, flavoring agents, binders, surface active agents,thickeners, lubricants, suspending agents, preservatives (includingantioxidants) and the like.

The active compounds described herein (including all isomers) and saltsthereof of the invention are intended to be administered under theguidance of a physician or veterinarian.

The amount of pharmacologically active compound (or any isomer thereof)or salt thereof required to be effective for antimicrobial treatmentwill, of course, vary with the individual mammal being treated and isultimately at the discretion of the medical or veterinary practitioner.The factors to be considered include the condition being treated, theroute of administration, the nature of the pharmaceutical composition,the mammal's body weight, surface area, age and general condition, andthe particular compound or salt to be administered. In general, thepharmaceutical compositions of this invention contain from about 0.5 toabout 500 mg and, preferably, from about 5 to about 350 mg of the activeingredient, preferably in a unit dosage form.

A suitable effective dose is in the range of about 0.1 to about 200mg/kg body weight per day, preferably in the range of about 1 to about100 mg/kg per day, calculated as the non-salt form of the activecompound. The total daily dose may be given as a single dose, multipledoses, e.g., two to six times per day, or by intravenous infusion for aselected duration. Dosages above or below the range cited above arewithin the scope of the present invention and may be administered to theindividual patient if desired and necessary.

For example, for a 75 kg mammal (preferably an human), a dose rangewould be about 7.5 to about 1500 mg per day, and a typical dose would beabout 800 mg per day.

If discrete multiple doses are indicated, treatment might typically be200 mg of a compound disclosed herein, given 4 times per day.

Topical Biocidal Compositions:

The subject compounds are also useful for sterilizing surfaces andrendering them resistant to subsequent cellular growth. Thus, forexample, one or more of the active ingredients disclosed herein,optionally in combination with a suitable carrier, can be used to treatthe surfaces of implantable medical devices prior to their implantation.For instance, pacemakers, stents, shunts, catheters, and the like can betreated with the compounds of the subject invention to render theseitems free of cellular contamination in general and bacterialcontamination in particular. Likewise, optical devices that are insertedinto the body, such as endoscopes, bronchoscopes, and the like, can alsobe treated with compounds of the subject invention, thereby renderingthe surface of the device microbe-free.

In practice, using the compounds of the present invention to treat asurface is a simple matter of contacting the surface with a sufficientamount of the active ingredient, for a time sufficient to allow thecompound to exert its biocidal effect. The active ingredient can beapplied neat, or in combination with a suitable carrier. Insofar as theactive ingredients will not be injected or ingested into the body, thecarrier for purposes of treating inanimate surfaces need not bepharmaceutically suitable.

Alternatively, the subject compounds can be covalently bonded to thesurface of interest. In one approach, the fully-formed compounds can beattached covalently using the same chemistries described herein. Also,the subject compounds can be synthesized directly attached to thesurface via covalent bonds by polymerizing the compounds de novo from aninitiator group that is covalently attached to the surface of interest.Again, this can be accomplished using the chemistries described herein.

Examples

The following Examples are included solely to provide a more clear andconsistent understanding of the invention disclosed and claimed herein.

Materials: Substituted styrenes (liquid) (obtained commercially fromMonomer-Polymer/Daj ac Laboratories. Feasterville, Pa.) were purified byvacuum distillation (representative boiling points: DMAS 1, 73° C. at1.7 mm Hg; 2, 122-125° C. at 1.8 mm Hg; 3, 42° C. at 0.75 mm Hg) andstored at −80° C. until use. Styrene 4 (Aldrich, Milwaukee, Wis.) waspurified by passage through an inhibitor-removal column (SDTR-7,Scientific Polymer Products, Ontario, New York) immediately before use.VBC (a 68:32 mixture of m- and p-isomers, Aldrich) was purified byvacuum distillation immediately before use. Benzene was distilled fromsodium benzophenone ketyl. Methanol was distilled from Mg(OMe)₂. AIBN,dodecyldimethylamine, and (ar-vinylbenzyl)trimethylammonium chloride 5(Aldrich) were used without further purification. Monomer 6 wassynthesized from VBC and dodecyldimethylamine by the procedure of Ikedaet al.¹⁶

General Procedure for Polymerizations and Stock Solutions: Monomer (2.00mmol total) was degassed by three freeze-pump-thaw cycles in a 25 mLSchlenck flask. AIBN was flushed with N₂ for 1 h and dissolved to 16.4mg/mL (0.1 M) in degassed benzene. A 1 mL aliquot of this solution wasadded to the Schlenck flask, which was heated to 60° C. with stirringfor 2 d. The reaction mixture was concentrated under centrifugation,resuspended in chloroform, applied to a column of Sephadex LH-20 (20 mmdiameter×75 mm length), eluted with chloroform, and collected as an 8 mLpre-run, 1-2 mL fractions, and a post-run. RP-TLC (Whatman LKC18Fplates, MeOH eluent, UV visualization) was used to check fractions forresidual monomer (R_(f)˜0.5). Fractions showing only the baselinepolymer streak were pooled and concentrated under centrifugation. Thisprocedure afforded each purified polymer as a clear oil.

Chloride salts of polymers were obtained by dissolving the polymers, invials, in 0.02 N HCl (prepared by dilution of sterile-filtered 1.0 N HCl(Sigma, St. Louis, Mo.) with Millipore water) to a concentration of 2mg/mL, then drying by centrifugal evaporation with heating to 80° C. for5-10 h. Millipore water was then added to the vials to give stocksolutions (2 mg/mL) of free-amine polymer (not accounting for the addedweight due to formation of the HCl salt). Poly(5) was directly solublein water. Poly(6) was first dissolved in 2:1 (v/v) DMF:ethanol to aconcentration of 33 mg/mL, in accordance with Senuma et al^(18, 19) andthen diluted with Millipore water to stock concentration of 2 mg/mL.This solution was allowed to stand overnight at room temperature; noprecipitation was observed. Solute-free DMF:ethanol was similarlydiluted to serve as an organic solvent blank. These stock solutions wereused in the Orange OT solubilization, ANS binding, antibacterialactivity, and hemolysis assays described herein.

Characterization Data for Polymers: The following polymers weresynthesized and purified as described above, and then characterized byvarious methods, including percent yield, elemental analysis, ¹H and ¹³CNMR, gel permeation chromatography (GPC, also known as size-exclusionchromatography), UV-Vis spectroscopy, and mass spectrometry. The resultswere as follows:

Poly(1): 17.7 mg (110 μmol, 5.5%). ¹H NMR δ (CDCl₃, 300 MHz) 0.887 ppm(lump, 0.17H), 1.066 ppm (lump, 0.14H), 1.362 ppm (lump, 1.79H,backbone), 1.673 ppm (lump, 1.11H, backbone), 2.154 ppm (double lump,6.08H, Me₂N), 2.677 ppm (lump, 0.36H), 3.266 ppm (br lump, 1.94H,ArCH₂N), 6.385 ppm (lump, 1.84H, aromatic), 6.91 ppm (lump, 2.12H,aromatic). ¹³C NMR δ (CDCl₃, 75 MHz) 45.247 ppm (negative to DEPT-135,NMe₂). UV/Vis (MeOH) 204 nm (ε=589200 cm²/g), 258 nm (ε=44800 cm²/g).GPC M_(w)=8.14 KDa, M_(n)=2.49 KDa, PDI=3.27.

Poly(1:2)3: 64.5 mg (396 μmol, 20%). ¹H NMR δ (CDCl₃, 300 Mhz) 0.9 ppm(lump, 0.27H, octyl methyl), 1.051 ppm (lump, 0.16H), 1.296 ppm (lump,2.16H, backbone, octyl), 1.652 ppm (lump, 1.18H, backbone), 2.152 ppm(double lump, 6.47H, Me₂N), 2.441 ppm (lump, 0.07H. ArCH₂ octyl), 3.242ppm (br lump, 1.91H, ArCH₂N), 6.393 ppm (lump, 1.82H, aromatic), 6.909ppm (lump, 2.14H, aromatic). GPC M_(w)=8.57 KDa, M_(n)=3.29 KDa,PDI=2.60.

Poly(1:2)5: 33.2 mg (202 μmol, 10%). ¹H NMR δ (CDCl₃, 300 MHz) 0.896 ppm(irregular lump, 0.33H, octyl methyl), 1.063 ppm (lump, 0.15H), 1.296ppm (lump, 2.37H, backbone, octyl), 1.587 ppm (lump, 1H, backbone), 2.08ppm (two lumps, 5.78H, Me₂N), 2.444 ppm (lump, 0.19H, octyl ArCH₂),3.291 ppm (br lump, 1.83H, ArCH₂N), 4.603 ppm (lump, 0.06H), 6.372 ppm(lump, 1.82H, aromatic), 6.883 ppm (lump, 2.18H, aromatic). GPCM_(w)=9.87 KDa, M_(n)=2.21 KDa, PDI=4.47.

Poly(1:2)7: 44.2 mg (268 μmol, 13%). ¹H NMR δ (CDCl₃, 300 Mhz) 0.896 ppm(lump, 0.45H, octyl methyl), 1.068 ppm (lump, 0.22H), 1.264 ppm (lump,2.38H, backbone, octyl), 1.763 ppm (lump, 2.82H, backbone), 2.164 ppm(double lump, 5.95H, Me₂N), 2.454 ppm (lump, 0.16H, ArCH₂ octyl), 3.233ppm (br lump, 1.89H, ArCH₂N), 6.386 ppm (lump, 1.7H, aromatic), 6.887ppm (lump, 2.23H, aromatic). UV/Vis (MeOH) 204 nm (ε=284900 cm²/g), 256nm (ε=23000 cm²/g). GPC M_(w)=11.0 KDa, M_(n)=2.94 KDa, PDI=3.75.

Poly(1:2)10: 37.3 mg (224 μmol, 11%). ¹H NMR δ (CDCl₃, 300 MHz) 0.882ppm (lump, 0.57H, octyl methyl), 1.294 ppm (lump, 2.88H, octyl,backbone), 1.657 ppm (multiple lumps, 2.63H, backbone, water), 2.139 ppm(two lumps, 5.87H, Me₂N), 2.475 ppm (lump, 0.27H, ArCH₂ octyl), 3.214ppm (two lumps, 2.05H, ArCH₂N), 6.407 ppm (lump, 1.63H, aromatic), 6.909ppm (lump, 2.36H, aromatic). ¹³C NMR δ (CDCl₃, 75 MHz) 44.923 ppm(NMe₂). UV/Vis (MeOH) 204 nm (ε=361100 cm²/g), 268 nm (ε=28500 cm²/g).GPC M_(w)=10.3 KDa, M_(n)=3.44 KDa, PDI=3.00.

Poly(1:2)15: 57.1. mg (337 μmol, 17%). ¹H NMR δ (CDCl₃, 300 MHz) 0.898ppm (lump, 0.86H, octyl methyl), 1.302 ppm (multiple lumps, 5.04H,backbone, octyl), 2.15 1 ppm (two lumps, 4.7H, Me₂N), 2.405 ppm (lump,1.44H, ArCH₂ octyl), 3.231 ppm (lump, 1.99H, ArCH₂N), 6.408 ppm (lump,1.77H, aromatic), 6.924 ppm (lump, 2.15H, aromatic). GPC M_(w)=5.74 KDa,M_(n)=2.85 KDa, PDI=2.01.

Poly(1:2)20: 26.9 mg (156 μmol, 7.8%). ¹H NMR δ (CDCl₃, 300 MHz) 0.896ppm (lump, 0.83H, octyl methyl), 1.295 ppm (lump, 3.72H, octyl,backbone), 1.673 ppm (multiple lumps, 2.26H, backbone, water), 2.148 ppm(two lumps, 4.99H, Me₂N), 2.486 ppm (lump, 0.46H, ArCH₂ octyl), 3.207ppm (two lumps, 1.67H, ArCH₂N), 6.407 ppm (lump, 1.75H, aromatic), 6.932ppm (lump, 2.23H, aromatic).

Poly(1:2)30: 7.40 mg (41.6 μmol, 2.1%). ‘H NMIR 8 (CDCl₃, 300 MHz) 0.895ppm (lump, 1.37H, octyl methyl), 1.298 ppm (multiple lumps, 6.96H,backbone, octyl), 2.164 ppm (two lumps, 4.39H, Me₂N), 2.466 ppm (lump,1.96H, ArCH₂ octyl), 3.303 Ppm (two lumps, 2.06H, ArCH₂N), 6.396 ppm(lump, 1.69H, aromatic), 6.93 ppm (lump, 2.25H, aromatic). GPCM_(w)=5.82 KDa, M_(n)=3.01 KDa, PDI=1.94.

Poly(1:2)35: 13.3 mg (73.7 μmol, 3.7%). ¹H NMR δ (CDCl₃, 300 MHz) 0.895ppm (lump, 1.29H, octyl methyl), 1.293 ppm (lump, 5.26H, octyl,backbone), 1.695 ppm (multiple lumps, 1.44H, backbone, water), 2.15 ppm(two lumps, 4.02H, Me₂N), 2.479 ppm (lump, 0.78H, ArCH₂ octyl), 3.225ppm (two lumps, 1.18H, ArCH₂N), 6.41 ppm (lump, 1.8H, aromatic), 6.909ppm (lump, 2. 17H, aromatic).

Poly(1:3)3: 71.6 mg (445 μmol, 22%). ¹³C NMR δ (CDCl₃, 75 MHz) 126.256ppm (broad, positive to DEPT-135, aromatic), 127.15 ppm (broad, positiveto DEPT-135, aromatic), 127.815 ppm (broad, positive to DEPT-135,aromatic), 128.535 ppm (broad, positive to DEPT-135, aromatic), 40.281ppm (positive to DEPT-135, backbone), 45.202 ppm (positive to DEPT-135,NMe₂), 64.056 ppm (negative to DEPT135, ArCH₂N), 64.249 ppm (negative toDEPT-135, ArCH₂N). ¹H NMR δ (CDCl₃, 300 MHz) 0.886 ppm (lump, 0.16H,backbone), 1.061 ppm (lump, 0.21H, backbone), 1.363 ppm (lump, 1.92H,backbone, iPr), 1.727 ppm (lump, 0.97H, backbone), 2.141 ppm (two lumps,5.89H, NMe₂), 3.285 ppm (double lump, 1.95H, ArCH₂N), 6.413 ppm (lump,1.75H, aromatic), 6.912 ppm (lump, 2.13H, aromatic). GPC M_(w)=7.55 KDa,M_(n)=2.56 KDa, PDI=2.95.

Poly(1:3)5: 22.6 mg (141 μmol, 7.0%). ¹H NMR δ (CDCl₃, 300 MHz) 0.888ppm (lump, 0.19H, backbone), 1.335 ppm (lump, 2.22H, backbone, iPr),1.664 ppm (lump, 0.97H, backbone), 2.146 ppm (two lumps, 5.94H, NMe₂),2.829 ppm (lump, 0.26H, iPr ArCH₂), 3.229 ppm (double lump, 1.82H,ArCH₂N), 6.382 ppm (lump, 1.85H, aromatic), 6.913 ppm (lump, 2.14H,aromatic). ¹³C NMR δ (CDCl₃, 75 IvIIlz) 127.621 ppm (lump, positive toDEPT-135, aromatic), 45.227 ppm (positive to DEPT-135, NMe₂), 64.268 ppm(lump, negative to DEPT-135, ArCH₂N). GPC M_(w)=8.63 KDa, M_(n)=3.35KDa, PDI=2.58.

Poly(1:3)7: 28.3 mg (177 μmol, 8.8%). ¹H NMR δ (CDCl₃, 300 Mhz) 0.891ppm (lump, 0.1H, backbone), 1.059 ppm (lump, 0.22H, backbone), 1.361 ppm(lump, 2.22H, backbone, iPr), 1.684 ppm (lump, 0.88H, backbone), 2.143ppm (two lumps, 5.98H, NMe₂), 3.217 ppm (double lump, 1.86H, ArCH₂N),6.391 ppm (lump, 1.85H, aromatic), 6.913 ppm (lump, 2.18H, aromatic).¹³C NMR δ (CDCl₃, 75 MHz) 126.384 ppm (positive to DEPT-135, aromatic),127.226 ppm (positive to DEPT-135, aromatic), 127.362 ppm (positive toDEPT-135, aromatic), 127.803 ppm (positive to DEPT-135, aromatic),128.484 ppm (positive to DEPT-135, aromatic), 128.571 ppm (positive toDEPT-135, aromatic), 40.236 ppm (positive to DEPT-135, iPr methyls),45.241 ppm (positive to DEPT-135, NMe₂), 64.085 ppm (negative toDEPT-135, ArCH₂N), 64.302 ppm (negative to DEPT-135, ArCH₂N). IR (thinfilm) 1032 cm⁻¹ (s), 1100 cm⁻¹ (w), 1148 cm⁻¹ (w), 1178 cm⁻¹ (m), 1264cm⁻¹ (m), 1368 cm⁻¹ (m), 1458 cm⁻¹ (s), 2770 cm⁻¹ (s), 2812 cm⁻¹ (s),2856 cm⁻¹ (w), 2932 cm⁻¹ (s), 712 cm⁻¹ (s), 794 cm⁻¹ (m), 816 cm⁻¹ (w),846 cm⁻¹ (m), 860 cm⁻¹ (m), 898 cm⁻¹ (w), 988 cm⁻¹ (w). UV-Vis (MeOH)204 nm (c=418400 cm²/g), 258 nm (c=30200 cm²/g). GPC M_(w)=7.45 KDa,M_(n)=2.86 KDa, PDI=2.61.

Poly(1:3)10: 17.3 mg (108 μmol, 5.4%). ¹H NMR δ (CDCl₃, 300 MHz) 0.817ppm (lump, 0.2H, backbone), 1.284 ppm (lump, 2.61H, backbone, iPr),1.669 ppm (lump, 0.97H, backbone), 2.153 ppm (two lumps, 5.78H, NMe₂),2.78 1 ppm (lump, 0.18H, iPr ArCH₂), 3.239 ppm (double lump, 1.7 1H,ArCH₂N), 6.404 ppm (lump, 1.86H, aromatic), 6.911 ppm (lump, 2.14H,aromatic). ¹³C NMR δ (CDCl₃, 75 MHz) 127.869 ppm (lump, positive toDEPT-135, aromatic), 45.178 ppm (positive to DEPT-135, NMe₂), 64.122 ppm(lump, negative to DEPT-135, ArCH₂N). GPC M_(w)=9.16 KDa, M_(n)=2.71KDa, PDI=3.38.

Poly(1:3)15: 31.8 mg (200 μmol, 10%). ¹H NMR δ (CDCl₃, 300 MHz) 0.894ppm (lump, 0.14H, backbone), 1.08 1 ppm (lump, 0.3 1H, backbone), 1.361ppm (lump, 2.57H, backbone, iPr), 1.684 ppm (lump, 0.93H, backbone),2.145 ppm (two lumps, 5.13H, NMe₂), 3.219 ppm (double lump, 1.69H.ArCH₂N), 6.406 ppm (lump, 1.78H, aromatic), 6.916 ppm (lump, 2.11H,aromatic). ¹³C NMR δ (CDCl₃, 75 MHz) 126.402 ppm (broad, positive toDEPT-135, aromatic), 127.304 ppm (broad, positive to DEPT-135,aromatic), 127.863 ppm (broad, positive to DEPT-135, aromatic), 128.62ppm (broad, positive to DEPT-135, aromatic), 40.237 ppm (positive toDEPT-135, backbone), 45.217 ppm (positive to DEPT-135, NMe₂), 64.094 ppm(negative to DEPT-135, ArCH₂N), 64.319 ppm (negative to DEPT135,ArCH₂N). GPC M_(w)=10.3 KDa, M_(n)=3.32 KDa, PDI=3.09.

Poly(1:3)20: 24.4 mg (154 μmol, 7.7%). ¹H NMR δ (CDCl₃, 300 Mhz) 0.86ppm (lump, 0.18H, backbone), 1.186 ppm (lump, 3.16H, backbone, iPr),1.706 ppm (lump, 0.93H, backbone), 2.153 ppm (two lumps, 5.06H, NMe₂),2.767 ppm (lump, 0.17H, iPr ArCH₂), 3.239 ppm (double lump, 1.66H,ArCH₂N), 6.404 ppm (lump, 1.84H, aromatic), 6.911 ppm (lump, 2.17H,aromatic). ¹³C NMR δ (CDCl₃, 75 Mhz) 127.23 ppm (lump, positive toDEPT-135, aromatic), 24.038 ppm (positive to DEPT-135, isopropyl),40.196 ppm (lump, positive to DF.PT-135, isopropyl), 45.19 ppm (positiveto DEPT-135, NMe₂), 64.051. ppm (negative to DEPT-135, ArCIHN₂), 64.272ppm (negative to DEPT-135, ArCHN₂). GPC M_(w)=9.50 KDa, M_(n)=3.26 KDa,PDI=2.91.

Poly(1:3)30: 104 mg (663 μmol, 33%). ¹H NMR δ (CDCl₃, 300 MHz) 0.896 ppm(lump, 0.22H, backbone), 1.186 ppm (overlapping lumps, 3.73H, backbone,isopropyl), 1.732 ppm (lump, 2H, backbone), 2.158 ppm (two lumps, 4.28H,Me₂N), 2.796 ppm (lump, 0.32H), 3.253 ppm (double lump, 1.39H, ArCH₂N),6.47 ppm (lump, 1.76H, aromatic), 6.92 ppm (lump, 2.24H, aromatic). GPCM_(w)=7.63 KDa, M_(n)=3.07 KDa, PDI=2.49.

Poly(1:4)3: 62.2 mg (390 μmol, 19%). ¹H NMR δ (CDCl₃, 300 MHz) 0.895 ppm(lump, 0.18H, backbone), 1.336 ppm (lump, 1.96H, backbone), 1.719 ppm(lump, 2.29H, backbone), 2.145 ppm (two lumps, 5.84H, Me₂N), 3.246 ppm(lump, 1.94H, ArCH₂N), 6.377 ppm (lump, 1.77H, aromatic), 6.899 ppm(lump, 2.24H, aromatic). GPC M_(w)=8.19 KDa, M_(n)=3.22 KDa, PDI=2.54.

Poly(1:4)5: 79.2 mg (500 μmol, 25%). ¹H NMR δ (CDCl₃, 300 MHz) 0.888 ppm(lump, 0.2H, backbone), 1.065 ppm (lump, 0.21H, backbone), 1.363 ppm(lump, 2.07H, backbone), 1.664 ppm (lump, 1.14H, backbone), 2.143 ppm(two lumps, 6.68H, NMe₂), 3.219 ppm (double lump, 2H, ArCH₂N), 6.382 ppm(lump, 2.13H, aromatic), 6.924 ppm (lump, 2.52H, aromatic). ¹³C NMR δ(CDCl₃, 75 MHz) 126.344 ppm (broad, positive to DEPT-135, aromatic),127.096 ppm (broad, positive to DEPT-135, aromatic), 127.816 ppm (broad,positive to DEPT-135, aromatic), 128.522 ppm (broad, positive toDEPT-135, aromatic), 40.189 ppm (positive to DEPT-135, backbone), 45.183ppm (positive to DEPT-135, NMe₂), 63.991 ppm (negative to DEPT-135,ArCH₂N), 64.232 ppm (negative to DEPT-135, ArCH₂N). GPC M_(w)=8.17 KDa,M_(n)=3.12 KDa, PDI=2.62.

Poly(1:4)7: 15.6 mg (99.2 μmol, 5.0%). ¹H NMR δ (CDCl₃, 300 Mhz) 0.895ppm (lump, 0.18H, backbone), 1.32 ppm (lump, 1.91H, backbone), 1.826 ppm(lump, 1.6H, backbone), 2.145 ppm (two lumps, 5.71H, Me₂N), 3.27 ppm(lump, 1.85H, ArCH₂N), 6.326 ppm (lump, 1.75H, aromatic), 6.865 ppm(lump, 2.25H, aromatic). ¹³C NMR 6 (CDCl₃, 75 MHz) 127.344 ppm (multiplepositive peaks on DEPT-135, aromatic), 45.285 ppm (positive to DEPT-135,NMe₂), 64.384 ppm (lumpy, negative to DEPT-135, ArCH₂N). UV/Vis (MeOH)204 nm (c=468000 cm²/g), 256 nm (c=26300 cm²/g). GPC M_(w)=4.56 KDa,M_(n)=1.85 KDa, PDI=2.47.

Poly(1:4)10: 44.0 mg (283 μmol, 14%). ¹H NMR δ (CDCl₃, 300 MHz) 0.896ppm (lump, 0.21H, backbone), 1.076 ppm (lump, 0.2H, backbone), 1.375 ppm(lump, 1.88H, backbone), 1.689 ppm (lump, 1.09H, backbone), 2.153 ppm(two lumps, 5.89H, NMe₂), 3.226 ppm (double lump, 2H, ArCH₂N), 6.408 ppm(lump, 1.85H, aromatic), 6.946 ppm (lump, 2.46H, aromatic). ¹³C NMR δ(CDCl₃, 75 Mhz) 127.83 ppm (broad, positive to DEPT-135, aromatic),40.325 ppm (positive to DEPT-135, backbone), 45.227 ppm (positive toDEPT-135, NMe₂), 64.015 ppm (negative to DEPT-135, ArCH₂N), 64.275 ppm(negative to DEPT-135, ArCH₂N). GPC M_(w)=8.19 KDa, M_(n)=3.17 KDa,PDI=2.58.

Poly(1:4)15: 18.4 mg (121 μmol, 6.0%). ¹H NMR δ (CDCl₃, 300 MHz) 0.897ppm (lump, 0.13H, backbone), 1.276 ppm (lump, 2.06H, backbone), 1.673ppm (lump, 3.36H, backbone), 2.157 ppm (two lumps, 5.1H, Me₂N), 3.27 ppm(lump, 1.82H, ArCH₂N), 6.377 ppm (lump, 1.7H, aromatic), 6.938 ppm(lump, 2.32H, aromatic). GPC M_(w)=9.82 KDa, M_(n)=3.21 KDa, PDI=3.06.

Poly(1:4)20: 54.6 mg (364 μmol, 18%). ¹H NMR δ (CDCl₃, 300 MHz) 0.884ppm (lump, 0.18H, backbone), 1.072 ppm (lump, 0.19H, backbone), 1.369ppm (lump, 2.1H, backbone), 1.7 ppm (lump, 1.19H, backbone), 2.149 ppm(two lumps, 6.11H, NMe₂), 3.281 ppm (double lump, 2H, ArCH₂N), 6.411 ppm(lump, 2.15H, aromatic), 6.951 ppm (lump, 2.87H, aromatic). ¹³C NMR δ(CDCl₃, 75 MHz) 126.402 ppm (broad, positive to DEPT-135, aromatic),127.304 ppm (broad, positive to DEPT-135, aromatic), 127.863 ppm (broad,positive to DEPT-135, aromatic), 128.62 ppm (broad, positive toDEPT-135, aromatic), 40.237 ppm (positive to DEPT-135, backbone), 45.217ppm (positive to DEPT-135, NMe₂), 64.094 ppm (negative to DEPT-135,ArCH₂N), 64.3 19 ppm (negative to DEPT-135, ArCH₂N). GPC M_(w)=8.84 KDa,M_(n)=3.41 KDa, PDI=2.59.

Poly(1:4)30: 36.8 mg (255 μmol, 13%). ¹H NMR δ (CDCl₃, 300 MHz) 0.905ppm (lump, 0.1H, backbone), 1.369 ppm (lump, 1.86H, backbone), 1.728 ppm(lump, 1.8H, backbone), 2.153 ppm (two lumps, 4.05H, Me₂N), 3.225 ppm(lump, 1.32H, ArCH₂N), 6.434 ppm (lump, 1.69H, aromatic), 6.955 ppm(lump, 2.31H, aromatic). GPC M_(w)=11.1 KDa, M_(n)=4.96 KDa, PDI=2.24.

TLC Assay for Copolymerization: A representative Sephadex-purifiedsample from each polymer series (poly(1:2)3, poly(1:3)7. andpoly(1:4)30) was spotted on Whatman K6F silica gel (60 Å particle size).As controls, pure poly(l) and polystyrene molecular weight standards (4KDa and a standard mixture with M_(p)=2,930 Da, 28.5 KDa, 148 KDa, 842KDa, and 7.50 MDa) were spotted onto the same plate. The plate waseluted with 1% (v/v) glacial acetic acid/ethyl acetate and visualized byUV. Poly(l) eluted as a streak with R_(f)<0.3; polystyrene standardseluted near the solvent front (R_(f)>0.9 for the 4 KDa sample, R_(f)>0.7for the standard mixture). All three experimental samples showed TLCpatterns-identical to poly(1), with no UV activity at Rf>0.3.

Determination of Apparent Molecular Weight: GPC was carried out atambient temperature (23-25° C.) using a Shimadzu HPLC system and pair(in series) of 300×7.5 mm PLgel Mixed D columns (5 μm pore size, PolymerLaboratories, Amherst, Mass.). Calibration was performed usingpolystyrene standards (EasiCal PS-1, Polymer Laboratories) of M_(p)841.7, 320, 148, 59.5, 28.5, 10.85, 2.93, and 0.58 KDa. Polymers weredissolved in eluent (0.1% TEA, freshly distilled from CaH₂, inHPLC-grade THF, Aldrich) to concentrations of ˜5 mg/mL, and 30 μLinjections were made. Ultraviolet absorbance data was collected usingShimadzu CLASS-VP software (version 7.1.1) and exported to MicrosoftExcel. A baseline was determined by linear regression analysis ofpeak-free regions and subtracted from the trace. The baseline-correctedtrace was then, integrated according to the method described by Yau etal.³⁰ to determine M_(w) and M_(n). Correction for peak spreading wasnot performed. PDI was calculated as M_(w)/M_(n).

Separate GPC analysis, performed on a representative subset of samplesusing a variety of PLgel columns using ELS detection, showed similarapparent MW. However, GPC performed on a Waters instrument in 2%NMP/THF, using a pair of Waters Styragel HT6E columns and RI detection,gave consistently larger apparent MW values (by approximately a factorof 3). This may be due to the 5 KDa lower detection limit of theStyragel columns and is mentioned as a caution that GPC-determinedmolecular weights are apparent.

Partial Fractionation: Samples of poly(1) and copoly(1₉₇:2₃), (1₉₅:2₅),(1₉₃:2₇), (1₉₀:2₁₀), (1₈₅:2₁₅), (1₈₀:2₂₀), and (1₇₀:2₃₀), were dissolvedin 0.2N HCl (prepared by dilution of sterile-filtered 1.0N HCl withMillipore water) to a concentration of 1-2 mg/mL One to ten mL (1-10 mL)of these solutions were added to Amicon Centriplus YM-50 filter units(Millipore, Bedford, Mass.), which had been rinsed once with

Millipore water. The filter units were centrifuged at 3000×g for 30 min.The retentates were collected by inverted spin retrieval. The filtrateswere then transferred to YM-30 filter units and centrifuged for 30 min.Further serial filtration through YM-10 (90 min) and YM-3 (250 min)filter units gave five fractions (i.e., the fractions retained by YM-50,retained by YM-30, retained by YM-10, retained by YM-3, and filtrate),which were dried by centrifugal evaporation. GPC analysis was performedas described above.

Dye Solubilization: Orange OT (Aldrich) was dissolved in ACSreagent-grade acetone, precipitated with Millipore water, recrystallizedtwice from absolute EtOH, and dried overnight under vacuum. Stocksolutions of polymers (2 mg/ml), prepared as described above, werediluted with Millipore water to give total volumes of 500 μL at theconcentration to be examined; mock dilutions of Millipore water wereperformed simultaneously to provide a separate blank for each polymerconcentration. Two identical vials of each polymer sample at eachconcentration were prepared; a small amount of Orange OT (less thanwould cover the tip of a small spatula) was added to one, and the other(treated identically but containing no Orange OT) was used as aspectroscopic blank to correct for absorbance of the polymer. All vialswere agitated gently for 3 d at room temperature on a blood-rocker andthen filtered through cotton to remove undissolved orange OT. An aliquot(200 μL) of each sample was diluted with 800 μL of absolute EtOH, andabsorbance of each solution was measured (1.0 cm path length cell, HP8452 UV/visible spectrometer) at 500 nm (Orange OT absorbance) and 340nm (background, to correct for baseline drift). Net absorbance(OD₅₀₀−OD₃₄₀), using the Orange OT-free samples as spectroscopic blanksand corrected for net absorbance of the polymer-free samples, wasdetermined.

ANS Fluorescence: A 10 mM stock solution of high-purity ANS (MolecularProbes, Eugene Oreg.) was prepared in Millipore water and transferred toa foil-wrapped vial. Vials were charged with 382.5 μL of 50 mM freshlyprepared Tris buffer (pH 6.8 at 23° C. in Millipore water, to avoid thepH dependence of ANS fluorescence, which arises below pH 6.0); 7.5 μL ofpolymer stock solutions (2 mg/mL), prepared as described above, orMillipore water (as a polymer-free blank) were added to pairs of vials.Ten μL of ANS stock solution was then added to one (1) vial from eachpair, and 10.0 μL of Millipore water to the other, to give 400 μL ofsolutions that were ˜250 μM in monomer residue and either 0 μM (ANS-freeblank) or 250 μM in ANS.

Fluorescence spectra were measured in a single session on a HitachiF-4500 fluorescence spectrophotometer. Excitation and emission slitwidths were set to 5 nm. Samples were excited at 350 nm; emissionspectra were collected from 370 nm to 650 nm. Polymer fluorescence inthe ANS-free blanks was of negligible intensity compared to thefluorescence of the ANS-containing samples, but the correspondingANS-free blank spectrum was subtracted from each ANS-containing samplespectrum.

Biological Properties:

MIC, MBC, and hemolysis assays were performed based upon standardprotocols, as described herein.

Minimal Inhibitory Concentration (MIC): Stock solutions of polymers(5.00 μL at 2 mg/mL, prepared as described above), commercial synthetic(Ala^(8,3,18))-1-magainin-2-amide (Sigma, dissolved in Millipore waterto 1 mg/mL, 10.0 μL, as positive control), or solvent blank (5.00 μL)were diluted into brain-heart infusion (BHI) broth in row A of 96-wellsterile assay plates (B-D Falcon 35-3075, Fisher Scientific) and sevenserial twofold dilutions were performed to give 50 μL of peptidesolution (at double the final assay concentration) in each well.Bacterial strains (Escherichia coli JM1O9, Bacillus subtilis BR151,²⁴Staphylococcus aureus 5332 (a clinical MRSA isolate from the Weisblumlaboratory strain collection), and Enterococcus faecium A436²⁵) weregrown on 2% bacteriological agar in BHI medium. Cells from thesecultures were suspended in BHI broth at a concentration of approximately10⁶ colony-forming units (CFU)/mL, and 50 μL of cell suspension wasadded to each well to give a total of 100 μL in each well.

Plates were then incubated at 37° C. for 6 h. Bacterial growth givesrise to turbidity and light scattering, which was detected by measuring0_(D650) using a Molecular Devices Emax microplate reader connected to aWindows computer running SOFTmax v.2.34. OD₆₅₀ values from twosimultaneous experiments were averaged. MIC, the concentration at whichgrowth is completely inhibited, is reported as the median value of atleast three experiments from separately diluted bacterial suspensions,at least two of which were performed on different days. If an evennumber of experiments was performed and the two median values were notidentical, the geometric mean is reported as the MIC.

Minimal Bactericidal Concentration (MBC): To determine cell viability,10 μL from each of the MIC, 2×MIC, and 4×MIC wells (up to the maximumconcentration of 50 μL/mL) was diluted 100-fold. Of this, 100 μL(containing at most 5×10² CFU) was plated on 2% bacteriological agar inBHI medium and incubated at 37° C. overnight (for B. subtilis) or for 4d (for E. coli, because no growth was seen after 10 h; colonies appearedon some plates after extended incubation). The absence of colonies wasconsidered indicative of >99% bacterial killing, and the lowest assaywell concentration showing no colonies is reported as the MBC.

Hemolysis Assay: Sterile TBS (Tris buffered saline: 10 mM Tris, 150 mMNaCl, pH 7.2) was prepared and used to dilute stock solutions ofpolymers (5.00 μL at 2 mg/mL prepared as described above), commercialsynthetic melittin (Sigma, dissolved in Millipore water to 1 mg/mL, 10.0μL, as positive control), or solvent blank (5.00 μL) in row A of 96-wellsterile assay plates. Seven serial twofold dilutions were performed togive 20 μL of peptide solution (at five times the final assayconcentration) in each well. Freshly drawn human red blood cells (hRBC,blood type A, collected into a Becton-Dickinson EDTA-anticoagulantvacuum container, refrigerated, and used within 8 h) were washed withTBS three times or until the supernatant was clear, then suspended inTBS at 1% (v/v). Eighty (80) μL of hRBC suspension was added to eachwell, giving a total assay volume of 100 μL. Plates were then incubatedat 37° C. for 1 h and centrifuged at 3500 rpm for 5 mm to pellet intacthRBC. Supernatant (80 μL) was diluted with 80 μL of twice-distilledwater and hemoglobin concentration measured (as OD₄₅₀) with a MolecularDevices Emax microplate reader. At this point, absorbance values wereaveraged across two identical plates. The most concentrated melittinwell in the plate (200 μL/mL or 50 μL/mL) was used as a reference for100% hemolysis; water blank was used as a reference for 0% hemolysis,and percent hemolysis was calculated as{OD₄₅₀(sample)−OD₄₅₀(buffer)}/{OD₄₅₀(melittin)−OD₄₅₀(buffer)}. The meanhemolysis percentage for a given polymer at a given concentration wascalculated, based on at least two separate experiments performed ondifferent days (except for poly(6), where two experiments were performedsimultaneously).

The results are presented in Tables 2 and 3:

TABLE 2 Biological Results for Various Polymers and Co-Polymers AgainstE. Coli and B. Subtilis: MBC Strain E. coli JM109 B. subtilis BR151Comonomer 2 3 4 2 3 4  0% [poly(1)] 50 50  3% >50 >50 >50 12.5 25 50  5%50  —a — 25 50 12.5  7% >50 50 >50 25 50 25 10% 50 — — 25 25 25 15% — —— 25 25 25 20% — — — >50 25 12.5 30% — — — >50 25 50 35% — — — >50 — —(Ala8,13,18)- 25 — magainin 2 amide aNot determined.

TABLE 3 Biological Results for Various Polymers and Co-Polymers AgainstE. Coli, B. Subtilis, S. aureus, and E Faecium: MIC Strain E. coli B.subtilis S. aureus E. faecium JM109 BR151 5332 A436 Comonomer 2 3 4 2 34 2 3 4 2 3 4  0% [poly(1)] 25 12.5 50 12.5  3% 50 25 25 12.5 6.3 8.8725 25 50 6.3 6.3 12.5  5% 50 25 50 12.5 8.87 35.4 25 25 >50 6.3 6.3 25 7% 25 25 25 6.3 6.3 8.87 12.5 50 50 3.2 6.3 6.3 10% 50 17.8 25 12.5 3.28.87 17.8 25 25 6.3 3.2 6.3 15% 50 25 25 25 8.87 12.5 25 25 25 12.5 6.312.5 20% 50 25 50 35.4 8.87 17.8 25  —a 25 25 — 12.5 30% >50 50 50 5017.8 25 >50 50 25 50 25 25 35% >50 — — >50 — — >50 — — 50 — —[Ala8,13,8]- 12.5 6.3 12.5 3.2 magainin 2 amide poly(5) >50 >50 >50 >50poly(6) >50 >50 >50 >50 water blank >50 >50 >50 >50 aq.DMF/EtOH >50 >50 >50 >50 1 (monomer) >50 >50 >50 >50 aNot determined.

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1-27. (canceled)
 28. A pharmaceutical composition for treating microbial infection in a subject in need thereof, the composition comprising an antimicrobial-effective amount of a compound selected from the group consisting of a polymer of formula -(A)_(n)-, wherein each A is a residue independently selected from the group consisting of:

wherein each R is independently selected from the group consisting of hydrogen; linear or branched C₁-C₃₀-alkyl, alkenyl, or alkynyl; unsubstituted amino-C₁-C₆-alkyl; mono-substituted amino-C₁-C₆-alkyl; disubstituted amino-C₁-C₆-alkyl; mono- or bicyclic aryl; mono- or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C₁-C₆-alkyl; and mono- or bicyclic heteroaryl-C₁-C₆-alkyl; wherein each R′ is independently selected from same group recited above for R, provided that one R′ is hydrogen; wherein n is an integer ≧3; and wherein at least one A is a residue containing a nitrogen atom, the nitrogen atom being capable of being quaternized, or a pharmaceutically suitable salt thereof.
 29. The pharmaceutical composition of claim 28, further comprising, in combination, a pharmaceutically suitable carrier.
 30. The pharmaceutical composition of claim 29, wherein the pharmaceutically suitable carrier is suitable for oral, rectal, parenteral, nasal or bronchial administration.
 31. The pharmaceutical composition of claim 28, wherein the compound has a molecular weight of between about 4.5 kDa and 11.5 kDa as determined by gel permeation chromatography.
 32. The pharmaceutical composition of claim 28, wherein each R is independently selected from the group consisting of hydrogen; linear or branched C₁-C₃₀-alkyl, alkenyl, or alkynyl; unsubstituted amino-C₁-C₆-alkyl; mono-substituted amino-C₁-C₆-alkyl; disubstituted amino-C₁-C₆-alkyl; mono- or bicyclic aryl; and mono- or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, and S.
 33. The method of claim 28, wherein n is an integer ≧6.
 34. A method of preventing and treating microbial infections in a subject in need thereof, the method comprising administering to the subject an effective anti-microbial amount of a compound selected from the group consisting of formula -(A)_(n)-, wherein each “A” is a residue independently selected from the group consisting of:

wherein each R is independently selected from the group consisting of hydrogen; linear or branched C₁-C₃₀-alkyl, alkenyl, or alkynyl; unsubstituted amino-C₁-C₆-alkyl; mono-substituted amino-C₁-C₆-alkyl; disubstituted amino-C₁-C₆-alkyl; mono- or bicyclic aryl; mono- or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C₁-C₆-alkyl; and mono- or bicyclic heteroaryl-C₁-C₆-alkyl; wherein each R′ is independently selected from same group recited above for R, provided that one R′ is hydrogen; wherein n is an integer ≧3; and wherein at least one A is a residue containing a nitrogen atom, the nitrogen atom being capable of being quaternized, or a pharmaceutically suitable salt thereof.
 35. The method of claim 34, wherein the compound is administered to a mammalian subject.
 36. The method of claim 34, wherein the compound is administered to a human subject.
 37. The method of claim 34, wherein the compound is administered in combination with a pharmaceutically suitable carrier.
 38. The method of claim 34, wherein the compound is administered in combination with a pharmaceutically suitable carrier suitable for oral, rectal, parenteral, nasal or bronchial administration.
 39. The method of claim 34, wherein a compound having a molecular weight of between about 4.5 kDa and 11.5 kDa as determined by gel permeation chromatography is administered to the subject.
 40. The method of claim 34, wherein a compound where each R is independently selected from the group consisting of hydrogen; linear or branched C₁-C₃₀-alkyl, alkenyl, or alkynyl; unsubstituted amino-C₁-C₆-alkyl; mono-substituted amino-C₁-C₆-alkyl; disubstituted amino-C₁-C₆-alkyl; mono- or bicyclic aryl; and mono- or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, and S is administered to the subject.
 41. The method of claim 34, wherein a compound wherein n is an integer ≧6 is administered to the subject.
 42. The method of claim 34, wherein the amount of compound administered to the subject is sufficient to provide a concentration of the compound, at point of contact with a microbial cell, of from about 1 μM to about 100 μM.
 43. The method of claim 34, wherein the amount of compound administered to the subject is sufficient to provide a concentration of the compound, at point of contact with a microbial cell, of from about 1 μM to about 10 μM. 